The present invention provides compositions and methods for diagnosing Alzheimer""s disease. In particular, the present invention provides modified beta-amyloid peptides, antibodies that specifically bind to the modified beta-amyloid peptides, and methods of use of these compositions in the diagnosis of Alzheimer""s disease, as well as methods to monitor treatment and/or disease progression of Alzheimer""s disease in patients. The present invention also provides compositions and methods useful in research involving amyloid precursor protein (APP) metabolism and Alzheimer""s disease.
Alzheimer""s disease (AD) is the fourth most common cause of death in the United States, next to heart disease, cancer and stroke. It presently afflicts more than four million people, and this number is expected to double during the next forty years with the aging of the population. Alzheimer""s disease is also the most common cause of chronic dementia, with approximately two million people in the United States suffering from dementia. At present, it is estimated that ten percent of the population older than 65 years of age have mild to severe dementia. This high prevalence, combined with the rate of growth of the elderly segment of the population, make dementia and particularly Alzheimer""s disease, important current public health problems.
To date, Alzheimer""s disease is the third most expensive disease in the United States, and costs approximately $100 billion each year. Costs associated with AD include direct medical costs such as nursing home care, direct nonmedical costs such as in-home day care, as well as indirect costs such as lost patient and care-giver productivity. Medical treatment may have economic benefits by slowing the rate of cognitive decline, delaying institutionalization, reducing care-giver hours, and improving quality of life.
Alzheimer""s disease is a complex multi-genic neurodegenerative disorder characterized by progressive impairments in memory, behavior, language, and visuo-spatial skills, ending ultimately in death. Hallmark pathologies of Alzheimer""s disease include granulovascular neuronal degeneration, extracellular neuritic plaques with xcex2-amyloid deposits, intracellular neurofibrillary tangles and neurofibrillary degeneration, synaptic loss, and extensive neuronal cell death. It is now known that these histopathologic lesions of Alzheimer""s disease correlate with the dementia observed in many elderly people.
Research on the causes of and treatments for Alzheimer""s disease has led investigators down numerous avenues. Although many models have been proposed, no single model of AD satisfactorily accounts for all neuropathologic findings; nor do these models of AD satisfactorily account for the requirement of aging for disease onset. Cellular changes, leading to neuronal loss and the underlying etiology of the disease, remain unknown. Proposed causes include environmental factors (Perl, Environmental Health Perspective 63:149 [1985]), metal toxicity (Perl et al., Science 208:297 [1980]), defects in beta-amyloid protein metabolism (Shijo et al., Science 258:126 [1992]; and Kosik, Science 256:780 [1992]), and abnormal calcium homeostasis and/or calcium activated kinases (Mattson et al., J. Neuroscience 12:376 [1992]). The mechanisms of disease progression are equally unclear. Considerable human genetic evidence has implicated alterations in production or processing of the human amyloid precursor protein (APP) in the etiology of the disease. However, intensive research has proven that AD is a multifactorial disease with many different, perhaps overlapping, etiologies.
Early detection and identification of Alzheimer""s disease facilitate prompt, appropriate treatment and care. However, there is currently no laboratory diagnostic test for Alzheimer""s disease. Although studies have suggested that calcium imaging measurement in fibroblasts were of potential clinical use in diagnosing Alzheimer""s disease (Peterson et al., Neurobiology of Aging 9:261 [1988]; and Peterson et al., Proc. Natl. Acad. Sci. USA 83:7999 [1986]), other studies using similar cell lines and techniques have shown no difference in calcium levels in Alzheimer""s and normal control fibroblasts (Borden et al., Neurobiology of Aging 13:33 [1991]). Thus, there remains a need for diagnostic methods for Alzheimer""s disease. In particular, reliable and cost-effective methods and compositions are needed to allow reliable diagnosis of Alzheimer""s disease.
The present invention provides compositions and methods for diagnosing Alzheimer""s disease. In particular, the present invention provides modified beta-amyloid peptides, antibodies that specifically bind to the modified beta-amyloid peptides, and methods of use of these compositions for the diagnosis of Alzheimer""s disease. The present invention also provides compositions and methods useful in research involving amyloid precursor protein (APP) metabolism and Alzheimer""s disease.
The present invention provides isolated and modified xcex2-amyloid (xcex2A) peptides comprising at least one malondialdehyde adduct. In one embodiment, the modified xcex2A peptide comprises the amino acid sequence of SEQ ID NO:1. In another embodiment, the modified xcex2A peptide comprises at least one malondialdehyde lysine adduct The present invention also provides purified antibodies directed against a modified xcex2-amyloid peptide comprising at least one malondialdehyde adduct. In some embodiments, the antibody is a monoclonal antibody, while in other embodiments, the antibody is a polyclonal antibody. In other related embodiments, the invention provides compositions comprising the antibody directed against the modified xcex2A-peptide comprising at least one malondialdehyde adduct.
The present invention further provides methods for detecting at least one modified xcex2-amyloid peptide comprising at least one malondialdehyde adduct, comprising the steps of: providing a sample suspected of containing at least one modified xcex2-amyloid peptide comprising at least one malondialdehyde adduct, and an antibody directed against at least on modified xcex2-amyloid peptide comprising at least on malondialdehyde adduct; contacting the sample with the antibody under conditions such that the antibody specifically binds to the modified xcex2-amyloid peptide comprising at least one malondialdehyde adduct, to form an antigen-antibody complex; and detecting the presence of the antigen-antibody complex. In some embodiments, the sample is selected from the group consisting of blood, plasma, serum, serous fluid, and cerebrospinal fluid. In some preferred embodiments, the sample is from a subject. In particularly preferred embodiments, the subject is a human. In further embodiments, the subject is selected from the group consisting of subjects displaying pathology resulting from Alzheimer""s disease, subjects suspected of displaying pathology resulting from Alzheimer""s disease, and subjects at risk of displaying pathology resulting from Alzheimer""s disease. In some particularly preferred embodiments, the methods further comprise the step of diagnosing Alzheimer""s disease. In additional particularly preferred embodiments, the Alzheimer""s disease diagnosed using the methods of the present invention is selected from the group consisting of late onset Alzheimer""s disease, early onset Alzheimer""s disease, familial Alzheimer""s disease and sporadic Alzheimer""s disease. In some preferred embodiments, the methods further comprise the step of monitoring the efficacy of treatment of Alzheimer""s disease.
In some preferred embodiments, the methods comprises an enzyme-linked immunosorbent assay. In particularly preferred embodiments, the enzyme-linked immunosorbent assay is selected from the group consisting of direct enzyme-linked immunosorbent assays, indirect enzyme-linked immunosorbent assays, direct sandwich enzyme-linked immunosorbent assays, indirect sandwich enzyme-linked immunosorbent assays, and competitive enzyme-linked immunosorbent assays. In alternative preferred embodiments, the antibody used in the methods of the present invention further comprises a conjugated enzyme, wherein the conjugated enzyme is selected from the group of enzymes consisting of horseradish peroxidases, alkaline phosphatases, ureases, glucoamylases, and xcex2-galactosidases. In some particularly preferred embodiments, the enzyme-linked immunosorbent assay further comprises an alkaline phosphatase amplification system. In alternative preferred embodiments, the methods further comprise at least one capture antibody, while in still further embodiments, the methods further comprise at least one detection antibody wherein the detection antibody is directed against the antibody directed against the modified xcex2-amyloid peptide comprising at least one malondialdehyde adduct. In still further embodiments, the detection antibody further comprises at least one conjugated enzyme selected from the group consisting of horseradish peroxidase, alkaline phosphatase, urease, glucoamylase and xcex2-galactosidase. In still further preferred embodiments, the methods further comprise the step of quantitating the at least one modified xcex2-amyloid peptide comprising at least one malondialdehyde adduct in the sample.
The present invention also provides kits for the detection of at least one modified xcex2-amyloid peptide comprising at least one malondialdehyde adduct, comprising an immobilized support and at least one antibody directed against at least one modified modified xcex2-amyloid peptide comprising at least one malondialdehyde adduct. In some preferred embodiments, the kit comprises an enzyme-linked immunosorbent assay kit. In still further embodiments, the kit further comprises components selected from the group consisting of needles, sample collection tubes, 96-well microtiter plates, instructions, at least one modified xcex2-amyloid peptide comprising at least one malondialdehyde adduct, an antibody-enzyme conjugate directed against a modified xcex2-amyloid peptide comprising at least one malondialdehyde adduct, at least one capture antibody, 96-well microtiter plates precoated with the at least one capture antibody, at least one coating buffer, at least one blocking buffer, distilled water, at least one enzyme-linked immunosorbent assay enzyme reaction substrate solution, and at least amplifier system. In some preferred embodiments, the amplifier system is an alkaline phosphatase enzyme-linked immunosorbent assay amplifier system.
The present invention also provides methods for detecting at least one antibody directed against a modified xcex2-amyloid peptide comprising at least one malondialdehyde adduct, comprising: a) providing a sample suspected of containing at least one antibody directed against a modified xcex2-amyloid peptide comprising at least one malondialdehyde adduct; at least one modified xcex2-amyloid peptide having at least one malondiadehyde adduct, and a detection antibody; b) contacting the sample with the modified xcex2-amyloid peptide, under conditions such that the antibody directed against a modified xcex2-amyloid peptide comprising at least one malondialdehyde adduct specifically binds to the modified xcex2-amyloid peptide to form an antigen-antibody complex; c) contacting the antigen-antibody complex with the detection antibody, under conditions such that the detection antibody specifically binds to the complex; and d) detecting the specific binding of the detection antibody to the antigen-antibody complex. In some preferred embodiments, the sample is selected from the group of samples consisting of blood, serous fluid, plasma, serum, cerebrospinal fluid, hybridoma conditioned culture medium, ascites fluid, and polyclonal antiserum. In some particularly preferred embodiments, the sample is from a subject, while in other preferred embodiments, the subject is human. In alternative preferred embodiments, the subject is selected from the group consisting of subjects displaying pathology resulting from Alzheimer""s disease, subjects suspected of displaying pathology resulting from Alzheimer""s disease, and subjects at risk of displaying pathology resulting from Alzheimer""s disease. In still further preferred embodiments, the mehtods further comprise diagnosing Alzheimer""s disease in the subject. In some preferred embodiments, the Alzheimer""s disease is selected from the group consisting of late onset Alzheimer""s disease, early onset Alzheimer""s disease, familial Alzheimer""s disease, and sporadic Alzheimer""s disease. In preferred embodiments, the method comprises an enzyme-linked immunosorbent assay. In some preferred embodiments, the enzyme-linked immunosorbent assay is selected from the group consisting of direct enzyme-linked immunosorbent assays, indirect enzyme-linked immunosorbent assays, direct sandwich enzyme-linked immunosorbent assays, indirect sandwich enzyme-linked immunosorbent assays, and competitive enzyme-linked immunosorbent assays. In still further embodiments, the detection antibody further comprises a conjugated enzyme, wherein the conjugated enzyme is selected from the group of enzymes consisting of horseradish peroxidases, alkaline phosphatases, ureases, glucoamylases, and xcex2-galactosidases. In additional embodiments, the enzyme-linked immunosorbent assay further comprises an alkaline phosphatase amplification system.
The present invention also provides kits for the detection of at least one antibody directed against at least one modified xcex2-amyloid peptide comprising at least one malondialdehyde adduct, comprising an immobilized support, at least one purified and modified xcex2-amyloid peptide comprising at least one malondialdehyde adduct, and at least one detection antibody. In some preferred embodiments, the kit is an enzyme-linked immunosorbent assay kit. In some preferred embodiments, the kit comprises components selected from the group consisting of needles, sample collection tubes, 96-well microtiter plates, instructions, at least one purified antibody directed against at least one xcex2-amyloid peptide comprising at least one malondialdehyde adduct, at least one 96-well microtiter plate precoated with at least one xcex2-amyloide peptide comprising at least one malondialdehyde adduct, at least one coating buffer, at least one blocking buffer, distilled water, at least one enzyme reaction substrate solution, and at least one amplifier system. In some particularly preferred embodiments, the amplifier system is an alkaline phosphatase enzyme-linked immunosorbent assay amplifier system.